(Adapted from Applicant's Abstract) Cell volume regulation is a necessary feature of human red cell (RBC) differentiation and in disease states with reticulocytosis and young cells. In sickle cell (SS), SC and CC disease, abnormal volume regulation leads to cell dehydration and pleiotropic pathological events. Unfortunately, RBC volume control is poorly understood, in particular, due to the lack of molecular identification of proteins responsible for volume regulation, and little understanding of the mechanisms by which these proteins are regulated. The major goals of this proposal are to molecularly define RBC volume- sensitive ion transporter/channel proteins and to understand the structural and regulatory responses these proteins undergo to restore normal volume. The investigators are particularly interested in defining the role of the cytoskeleton in transducing volume signals to the ion transporters/channel and whether direct interactions between cytoskeleton proteins and these proteins are involved int heir regulation. These goals will be addressed by the following specific aims: (1) Molecularly identify proteins responsible for RBC volume-sensitive ion transport: pICln, K:C1 co-transporter (K:C1-CT), and Na-K-C1 co-transporter (Na-K-2C1-CT); and crete cell and animal models expressing these ion transporters/channels. (2) Define the role of band 3 oligomeric state in volume-sensitive signal transduction and regulation of ion transporter/channel activity, and test the hypothesis that protein phosphorylation-dephosphorylation is a common mechanism for ion transport/channel regulation. If so, identify the phosphorylation sites. (3) Test the hypothesis that hemoglobin (Hb) binding to ion transport proteins/channels affects and cytoskeleton proteins. Define mechanisms by which cytoskeleton regulation of cell volume is affected. (5) Formulate models from this information, and test the models in erythroid cell lines where specific cytoskeleton deficiencies are induced using ribozyme and antisense technology. The studies should greatly increase the understanding the RBC volume regulation. This information will allow for sensible strategies aimed at preventing RBC dehydration, which would have a potentially significant impact on future drug or gene-based therapies for SS, SC, and CC diseases.